Creation Of Resistant Silkworm By Editing BmNPV Iap2 And Orf59

As a lepidoptera insect with important economic value,the silkworm is endangered by silkworm disease every year,which seriously restricts the sustainable development of silk industry of our country.Among them,the silkworm hematopoietic caused by Bombyx mori nucleopolyhedrovirus?BmNPV?is the most common and most harmful silkworm disease.Because the cycle of traditional resistance breeding is too long,and the resistance is more negatively related to economic traits,it is urgently needed to improve the disease resistance of silkworm by molecular breeding.In this study,we first knocked out the BmNPV apoptotic inhibitor iap2 and the proliferative replication related gene orf59 at the cellular level to detect its effect on the proliferation and replication of BmNPV.Transgenic silkworms were constructed by transgenic technology to edit BmNPV iap2 and orf59,and systematically tested the gene editing efficiency and off-target efficiency of the transgenic silkworm.Identifying the resistance level against BmNPV,analyzes the copy number of BmNPV and transcription level of different viral genes between mock and transgenic silkworm,and identification of transgenic silkworm growth status and economic characters;Finally,transcriptome data was used to analyze the genes of transgenic silkworm and analyze its resistance mechanism.The main results obtained of this thesis are as follows:1.Effect of BmNPV iap2 and orf59 on proliferation of BmNPVAn expression vector knocking out BmNPV iap2 and orf59 alone was constructed using the CRISPR/Cas9 system.qRT-PCR detection of viral transcription levels at different stages of the virus revealed viral gene expression was significantly downregulated at 48h by knockout of BmNPV iap2 or orf59.Further knocking out BmNPV iap2 and orf59 simultaneously,qRT-PCR analysis showed that the viral genes transcription level was further down-regulated,and at the same time the host apoptosis-related genes were detected after knockout of BmNPV iap2,and it was found that the expression level of the caspase family gene BmICE was significantly increased,and no significant change in the expression of the Bcl-2 family gene BmBuffy compared to controls.The above results show that,the apoptosis pathway of silkworm cells could be induced by knockout of BmNPV anti-apoptosis gene iap2 and the replication related gene orf59,thus inhibit BmNPV proliferation and replication and application of silkworm disease resistance breeding research2.Creation of the resistant materials to edit BmNPV iap2 and orf59.In order to further analyze the effect of anti-apoptosis to improve the antiviral effect of silkworm,the transgenic vector piggyBac[U6-sgIAP2-U6-sgORF59-3×P3DsRed afm]was successfully constructed.The transgenic silkworm of sgIAP2&sg ORF59 was obtained by microinjection technique.The sgIAP2&sgORF59 was crossed with a transgenic silkworm which expressing Cas9 protein to obtain the double positive strains Cas9?+?/sgIAP2&sgORF59?+?and the control lines Cas9?+?/sgIAP2&sg ORF59?-?,Cas9?-?/sgIAP2&sgORF59?+?,and Cas9?-?/sgIAP2&sgORF59?-?.Through T7E1 digestion and T-clone sequencing,it was found that double-positive strains can effectively edit target sites at 12h after the addition of BmNPV,and the editing efficiency reached 100%at 48h and continued until 120h.The statistical results of the editing frequency of the target genes showed that a large proportion of small fragments disappeared from the time of addition of the virus was only 19.4%at 12h,and the proportion of large fragment deletions reached 62.5%at 120h.The off-target sites with the highest off-target rate predicted by the two target sites were not detected by T-clone sequencing analysis.The above research shows that we have obtained the genetically modified gene editing silkworm which can specifically edit BmNPV genome.3.The resistance identification of transgenic silkworm and analysis of main economic charactersThe resistance levels of the two silkworm strains to BmNPV were determined by adding different doses of BmNPV to double-positive and control lines at 4th instar.The results showed that the onset time of Cas9?+?/sgIAP2&sgORF59?+?compared to Cas9?-?/sgIAP2&sgORF59?-?after adding different doses of ODV delayed by 1 to 2days,and the final mortality was higher than that of Cas9?-?/sgIAP2&sgORF59?-?.SPSS software further analyzed the LD₅₀ of the Cas9?+?/sgIAP2&sgORF59?+?silkworm was 2.25×10⁶polyhedron/head,compared to the control strain Cas9?-?/sgI AP2&sgORF59?-?which the LD₅₀ was 2.46×10⁴polyhedron/head,its resistance increased nearly 100-fold.The number of copies of viral DNA was detected by qRT-PCR,the results showed that the viral DNA copy number of double-positive strain was was significantly lower than control lines from 72h in the 4th instar,and the DNA copy number of the double positive strain was significantly inhibited from 96 h after the addition of BmNPV at the fifth instar.qRT-PCR showed that the virus genes of ie-1,gp64,vp39 and poly of double positive strains was significantly lower than that of the control line from 48h,and the transcription level of the viral gene was significantly lower from 96h after the 5th instar.In order to analyze whether the individual development and economic traits of the transgenic lines were affected,larvae and cocoon weight,cocoon shell rate and quantity of the cocoon layer weighing of four kinds of hybrid strains were statistical analysis,the results showed that there were no significant changes.The above results show that the transgenic silkworm can not only increase the resistance to BmNPV,but also have no effect on its economic character.4.Analysis of differential resistant genes of transgenic silkwormIn order to explore the resistance mechanism of transgenic silkworms,this study analyzed the transcriptional data of Cas9?+?/sgIAP2&sgORF59?+?and Cas9?-?/sgIA P2&sgORF59?-?after infected BmNPV 48h.Firstly,the virus DNA replication level of the extracted samples was detected in this study.The results showed that the expression level of vp39 in Cas9?+?/sgIAP2&sgORF59?+?strain was significantly lower than that of Cas9?-?/sgIAP2&sgORF59?-?after 48h of BmNPV infection.Transcriptome analysis showed that there were 709 differentially expressed genes,including 194 up-regulated genes and 515 down-regulated genes.The results showed that the differentially expressed genes were mainly concentrated in energy production and conversion,translation,ribosomal stracture and biogenesis,posttranslation modidification,protein turnover,chaperones.The differentially expressed genes were analyzed by KEGG pathway clustering,and the results showed that the differentially expressed genes were mainly concentrated in the oxidative phosphorylation pathway,with 41 differentially expressed genes,all of which were down-regulated expression genes.From these 41differentially expressed genes,12 genes were selected for qRT-PCR,and the results showed that the transcription level of transgenic silkworm was lower than Cas9?-?/sgIAP2&sgORF59?-?after infection of BmNPV 48h.and the results were consistent with the transcriptome data at the cellular level To identify the effect of BmNPV iap2 gene on apoptosis of silkworm cells,The transcriptional level of apoptosis-related genes Bmiap2,BmICE and BmBuffy were examined,the results showed that the expression of Bmiap2 was down-regulated and BmICE was up-regulated,BmBuffy expression was not significantly changed.It is indicated that after the knockout of BmNPV iap2 and orf59,it mainly causes the changes of the host energy level and apoptosis related pathways.